PROTON NMR SPECTROSCOPY OF HUMAN BLOOD Dallas L. Rabensteln, Kevin K. MilB, and Erin J. Sbauss D e p m e n t 01 Chemistry University of California Rivetside, CA 92521
NMR spectroscopy is an important technique for the study of biological fluids and intact cells (14). Compared with other analytical methods, it is nondestructive and noninvasive, and it allows delicately balanced chemical and cellular processes to be observed directly at the molecular level. 13C,'5N, 31P, and 'H NMR spectroscopies have all been used to study biological fluids andlor intact cells. Because of the inherent low NMR sensitivities and low natural ahundances of 13C and l5N, isotopically enriched compounds generally are used in 13C and '5N NMR studies. The advantage is a relatively simple spectrum that consists of resonancesfrom the enriched compounds and their metabolites superimposed on much weaker background resonances. However, isotopically enriched compounds are required, and, in the case of intact cell studies, they must be incorporated into the cells. Nevertheless, "C and 15N NMR with isotopically enriched compounds are widely used and important methods for the study of metabolic 31P, which has a natural abundance of loo90 and a high inherent NMR sensitivity, is also widely used. Because there are few pbosphorus-containing compounds at detectable levels, 31P
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NMR spectra of biological fluids and intact cells generally are relatively simple. For the same reason, however, 31P NMR can be used t o study only a limited number of compounds. Virtually all compounds in biological fluids and cells contain hydrogen, but the very abundance of bydrogen-containing compounds makes their study by 'H NMR more difficult. The measurement of 'H NMR spectra is further complicated by the water resonance. which obscures a large portion of the
spectrum and creates a dynamic range problem during data acquisition. However, because of its much greater sensitivity and the ubiquity of hydrogen in biological molecules, 'H NMR offers significant advantages. With state-ofthe-art high-field NMR spectrometers and some simple NMR experiments (6, 9), high-resolution 'H NMR spectra can he measured routinely for biological fluids and intact cells, particularly red blood cells. The purpose of this article is to re-
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FIgura 1. A 5 0 W H Z 'H NMR spectrum of human plasma. me spsnrum WBI~ m u r e d by mS slngls-pulae mmhod uslw a llip anple cd -5O
to avoid overioadlnp mS maloglc-dlpllal convew. This rpecrmm a d all tht, oher spectra preMnldIn ihls arlkle w s n m w e d wldl a Verlan VXR-SOOS sp&onwlw o w l e d in ms unlocked modn.
ANALYTICAL CHEMISTRY, VOL. 60, NO. 24. DECEMBER 15. IS88
0003-2700/88/A360-1380/SO 1.SO10 0 1988 Amerlcan Chemical Society
CELLS view some of the NMR techniques used to measure 'H NMR spectra of human plasma and red blood cells. The 'H NMR spectroscopy of plasma and red blood cells is of interest because reported studies indicate that information relevant to biochemical and clinical applications can be obtained by 'H NMR. 'H NMfl s p e d m m p y of plasma A 500-MHz 'H NMR spectrum of human plasma is shown in Figure 1. The
spectrum was measured by the standard single-pulse method and is characterized by a large water resonance at 4.11 ppm superimposed on a broad envelope of overlapping resonances. The broad envelope is primarily from plasma proteins and lipoproteins. Also superimposed on the broad envelope are
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much weaker resonances from small molecules in plasma (e.g., glucose) and resonances from the CH2 and CH3 groups of "mobile" fatty acid components of chylomicrons and lipoproteins. The water resonance blocks out a large and important part of the spectrum, and because the water proton concentration is -100 M compared with millimolar concentrations for the small molecules, it is difficult to detect the much weaker resonances. Thus it is desirable to eliminate the water resonance. Because this problem occurs in the measurement of 'H NMR spectra of most aqueous samples, a variety of water suppression methods have been developed (IO).These methods include suppression by presaturation of the water resonance, suppression by use of selective excitation pulse sequences (i.e., pulse sequences that selectively excite the spectral region of interest), and suppression by taking advantage of differences between the spin-lattice ( T I )or spin-spin (7'2) relaxation times of the water protons and those of the molecules of interest. Several of these methods have been used successfully to suppress the water resonance in 'H NMR spectra of plasma. Suppression by presaturation of the water resonance. Figure 2 shows the spectrum obtained from the same plasma sample with suppression of the water resonance by presaturation
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lgure 3. Schematic representations of the pulse sequences used to measure 1 NMR speclra of blood plasma and red blood cells. ilh mese pulse sequences, me water resnnance andla the broad envelope of r e m n c e s can be s u p essed In A and 8. '€" represents a flip angle 1.3 s), other resonances are also considerably reduced in intensity by spin-spin relaxation. Alternatively, several other approaches can he used to reduce the intensity of the water resonance. For example, the water resonance can be suppressed by selective saturation followed hy measurement of the spectrum by the CPMG pulse sequence (Ein Figure 3). With this method, a much shorter spin-spin relaxation period can be used. For example, the spectrum in
ANALYTICAL CHEMISTRY, VOL. 80. NO. 24, DECEMBER 15. is88
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'igure 9 was measured w i t h a spinpin relaxation period of 0.05 s. The idvantage is that relative resonance inensities are n o t so distorted by differ,ntial spin-spin relaxation (compare h e relative intensities of the phosphoipid-CH2and CH3 resonances and h e a l , L l , a n d v l resonancesinFigures I and 9). Alternatively, the spin-spin relaxition t i m e of the water protons can b e ,electively decreased by adding paranagnetic ions (25) or water proton ex:hange reagents (1 7,26,27) t o the sam)le. For example, addition of NH&l lecreases the water proton spin-spin ,elaxation time as a result of exchange
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of protons between ammonium ions and water molecules (26, 27). This method of water suppression, called the WATR method for Water Attenuat i o n b y TzRelaxation (26),allows resonances w i t h exactly the same chemical shift as the water resonance to be observed-an advantage over the presaturation method. T h e spectrum shown in Figure 10 was measured by the WATR method. 'H NMR s p e 3 m m p y ot rad blood cells T h e measurement o f high-resolution 'H-NMR spectra of intact r e d blood cells is complicated by the same fea-
.
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NMR spectrum of human plasma. Rrt qecbum was measured with pulse seqwnco E in Figure 3, using a T = 3.2 X 1 0 P s and 2 m = start 01 the CPMG 3.05 s. The demupler was on for water suppressionduring a period 01 1 I before pulse sequence and during Um pulse sequence; 128 transienls were co-added.
Figure 0. A portion of the 500-MHz CPMG
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I I Fburo 10. A Dortion of the 500-MHz CPMG 'H NMR spectrum of human plasma.
Sufficient NH&i was edded m give a concentration Of 0.5 M. and the pH was buffered at 7.1 wim phoSphate. The water resonancewas compietely snd selectively eliminated by lhe WATR m e W (Water Attenuation by T, Relaxation)using pulse sequence D In Figve 3 with 7 = 3.2 X lO-'s and 2 m = 0.5 s.
ANALYTICAL CHEMISTRY, VOL. 60. NO. 24, DECEMBER 15. 1988
tures as those for plasma (see the 500MHz IH NMR spectrum for packed red blood cells in Figure 11). The large water resonance is superimposed on a broad envelope of resonances, in this case from the protons of hemoglobin and the red cell membrane. The techniques discussed above can be used to selectively eliminate the interfering resonances and obtain high-resolution 'H NMR spectra for the small intracellular compounds (6,9). To illustrate, a 500-MHz spin-echo 'H NMR spectrum of intact red blood cells is shown in Figure 12. This spectrum was measured with a spin-spin relaxation period of 0.134 s. The hemoglobin and membrane resonances are eliminated, and the water resonance is significantly reduced in intensity. By
increasing the length of the spin-spin relaxation period, the water resonance can be further reduced in intensity (6, 28). Most of the resonances in the spinecho spectrum have been assigned to small intracellular compounds; the assignments are given in Figure 4. The spin-echo method has been used extensively in 'H NMR studies of chemical and metabolic processes in intact red blood cells. Figure 13 shows a 500-MHz CPMG 'H NMR spectrum of intact red blood cells. This spectrum, which is for a different sample of red blood cells, was measured with a longer spin-spin relaxation period (0.27 s) to completely eliminate the water resonance. Compared with the spin-echo spectrum in Figure 12,the resonances in this spec-
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Figure 11. The 500-MHz 'H NMR spectrum 01 numan rea DIW cells. The specrmm was measured by the single-pulse methcd using a flip angle of -5'. Sample preparation involved separating the red blood cells from plasma and white cells by cenblmgatlm.
Flgure 12. A portion of the 500-MHz spin-echo 'H NMR spectrum of intact red blood
cells. The SpeCtNm was measured with pulse sBquBncB C in Figure 3, using T = 0.007 6; 128 bansienls were cpBdded. The red b i d ceib used in the measurement of this spectrum were washed once with an equal
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YOlUme of i6otonic saline solution after sepration horn the plasma. To obtain @ resolution. the biwd was bubbled with oxygen to ensure that the hemoglobln was in the diamagnetic oxygenatad form.
ANALYTICAL CHEMISTRY, VOL. 60, NO. 24, DECEMBER 15, 1988
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trum are not phase modulated. Consequently the multiplet patterns are more easily recognized, and more resonances can be observed in the CPMG spectrum (e.g., the broad, relatively weak multiplet patterns in the 1.7-2.1ppm region). Some of the powerful two-dimensional 'H NMR techniques can also be applied to red blood cells by incorporating a spin-spin relaxation period in the pulse sequence to eliminate broad interfering resonances (29,30).For example, 'H COSY spectra have been measured for human and sheep red blood cells with the delayed COSY experiment (29). Resonance connectivities established from the COSY spectra were used to assign resonances in the 'H NMR spectrum of transport-deficient sheep red blood cells and to establish the presence of compounds whose resonances were hidden by overlap in one-dimensional spectra. Experimental conslderaUons The spectra in the figures were obtained with pulse sequences that generally are included in the standard software supplied with state-of-the-art pulseIFourier transform NMR spectrometers. However, several experimental considerations should be mentioned. 'H NMR methods for the noninvasive study of metabolism and other processes involving small molecules in intact red blood cells have also been described in detail elsewhere (6). Normally, NMR spectrometers are operated in a "locked" mode, using a lock signal derived from a deuterium resonance to maintain fieldfrequency stability. Because plasma and red blood cells do not contain sufficient natural-abundance deuterium for a lock signal, either D2O must be added to the sample or the spectrometer must be run in the unlocked mode. Fortu-
nately, the superconducting magnets in many high-field spectrometers are sufficiently stable, or, as is the case with the spectrometer used to measure the spectra presented here, they are equipped with drift correction hardware so that high-resolution spectra can be obtained without a fieldfrequency lock, even when signal averaging is required to achieve an adequate signal-to-noiseratio (S/N). The deuterium field/frequency lock signal is also generally used as a measure of magnetic field homogeneity when shimming the magnet. However, as long as sample volumes are carefully reproduced, we can obtain high-resolution spectra for plasma and red blood cells by using the same shim settings that give good spectra for a DzO solution of amino acids. Thus 'H NMR spectra can be obtained for intact plasma or red blood cells, without the need to add D20 or other reagents. Sample heatimg can occur when measuring spectra of plasma and red blood cells by the CPMG pulse sequence. Sample heating can affect the quality of the spectrum if the spectrometer is run in the locked mode. The effect of the sample heating on the quality of the spectrum can be eliminated by running an appropriate number of dummy scans to achieve a steady-state sample temperature before starting data acquisition or by running in the unlocked mode. Another important consideration is that it is advantageous to make measwements of the type described here on the highest field spectrometer available, not only for greater dispersion and sensitivity, but also because the spin-spin relaxation rate of HzO increases as the field strength increases because there is an exchange contribution to the spin-spin relaxation rate of H20 protons in protein solutions and
cell suspensions. The larger the field, the shorter the relaxation delay needed for elimination of the water resonance. The spectra presented in Figures 8,10, 12, and 13 demonstrate that at 500 MHz the water resonance can be selecbively eliminated with a spin-spin relaxation period sufficiently short that a good S/N is obtained for resonances from the low molecular weight compounds. With the high sensitivity of state-ofthe-art spectrometers, spectra of the type presented here can be obtained quickly, making it possible to follow relatively fast chemical processes (e.g., metabolic reactions) as a function of time directly in intact cells. With our spectrometer a reasonable 500-MHz CPMG 'H NMR spectrum of intact red blood cells can be obtained from four transients, which takes a total instrument time of 8 8. To achieve this high sensitivity, however, some basic data acquisition considerations should be kept in mind. With the large computer memory of state-of-the-art spectrometers, it may seem desirable to acquire large free induction decays (FIDs). However, acquisition of a large FIDs can have a deleterious effect on the spectrum, as illustrated by the series of spectra in Figure 14. These spectra are for the methyl protons of alanine and lactate in intact red blood cells. They were measured by the CPMG pulse sequence with a spin-spin relaxation period of 0.27 s. Each spectrum was obtained from four transients, no resolution or Sensitivity enhancement was applied, and each FID was zero-filled as necessary to give a total of 32,768 points before Fourier transformation. This series of spectra shows the effect of the length of data acquisition on the resolution and S/N.Acquisition of data for 0.103 8, which corresponds to the
Figure 13. A portion of the 500-MHz CPMG 'H NMR spectrum of intact red blood cells. The SPeCtNm w88 measured with pulse seqUBnCe D in Flgure 3. using T = 3.2 X I O P s and Z m = 0.27 s: 200 transients were co-added. The red b i d cells used In this meawement were not washed anw separation from p i a m .
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ANALYTICAL CHEMISTRY, VOL. 60, NO. 24, DECEMBER 15, 1988
Figure 14. Dependence of line shape and sensitivity on number of data points acquired and thus on the length of data acquisition. The data acquisition time8 were (fram lefl to right): 0.103, 0.205. 0.110. 0.819, 1.638and 3.277 8; four transiems were collected lor each spectrum. and FlDs were zero.lilled 88 nBoBSsary to give a total 01 32,788 paints belwe Fourier transformation: no resolution or sensitivity enhancement was applied. The resorimes are a1 (lefl)and L1 (rim)01 alanine and lactate. r~~pectlwly: the spectra were measured l a the red blood cell sample described in me legend of Flgure 13.
1024 point FID, is not long enough, as indicated by the truncation effects at the base of the lactate resonance. The truncation effects are eliminated by doubling the number of data points and thus the length of data acquisition. However, as the length of data acquisition is increased further, there is a decrease in SliV because, after a certain length of data acquisition, only noise is being acquired. Applicath The spectra in the figures demonstrate that with simple NMR experiments, high-resolution 'H NMR spectra can easily be obtained for small molecules in human plasma and red blood cells. A major disadvantage of 'H NMR is its low sensitivity; detection limits for small molecules in plasma and erythrocytes are on the order of 0.01-0.1 mmol/L, depending on the number of equivalent protons giving the resonance and its multiplicity. However, 'H NMR offers several advantages for clinical and biochemical studies, as illustrated by the following examples. Most of the reported 'H NMR studies of human plasma have been by Sadler, Nicholson, and co-worker8 (1317). They have assigned many of the resonances to the protons of mobile, low molecular weight metabolites in plasma, serum, and urine samples from fasting and diabetic subjects, including resonances for the ketone bodies 3-Dhydroxybutyrate, acetone, and aceto-
acetate (14). In a particularly interesting study that demonstrates the potential of high-resolution 'H NMR for clinical applications, 'H NMR spectra were measured for plasma and urine from subjects who had taken acetaminophen as a therapeutic dose or in self-poisoning episodes (both fatal and nonfatal) (16). The 'H NMR spectra of plasma from overdose patients suffering from acute liver failure showed gross elevation of lactate and the amino acids alanine, glutamine, proline, valine, methionine, serine, lysine, phenylalanine, tyrosine, and histidine. The patterns of plasma metabolite concentrations indicated by 'H NMR reflected characteristic and severe liver dysfunction, suggesting that 'H NMR spectroscopy of body fluids may be valuable in cases of drug overdose and perhaps in the diagnosis of various other diseases. For such applications, the advantages of NMR are that the spectrum can be obtained quickly and a wide range of metabolites present at millimolar concentrations often can be detected simultaneously rather than individually or in selected classes, as with conventional methods of clinical analysis. This capability was used in the diagnosis of D-lactic acidosis in a patient with jejuno-ileal bypass who developed a neurological syndrome associated with metabolic acidosis (31). L-lactate, as determined by an enzymatic procedure, was only 2.2 mmol/L, whereas 'H NMR gave a total lactate
(D- and L-lactate are indistinguishable by 'H NMR) concentration of 10 mmol/L. The diagnosis of D-lactic acidosis was subsequently confumed by a specific enzyme measurement of D-lactate, which gave a concentration of 7.5 mmoVL. A report that the average widths of the resonances for the CH2 and CH8 groups of mobile fatty acid components of lipoproteins in the 0.a1.3-ppm region correlated with the presence of malignant tumors in human subjects (32)stimulated considerable interest in the use of 'H NMR for the diagnosis of cancer. It was reported that the average width of the CH2 and CH3 resonances was 29.9 i 2.5 Hz in the presence of and 39.5 i 1.6 Hz in the absence of malignant tumors. A subsequent detailed study of these resonances showed that each contained several overlapping components from chylomicrons, very low density lipoprotein (VLDL), low-density lipoprotein (LDL), and high-density lipoprotein (HDL) (15).It also was found that average line widths of s.Bioeng. 1981. IO. 151 74. 18) Hobertr. J.K.M.;Jsrdetzk,,O. B u h i r n . B i o p h j s . Aero. 1981.6&.5R-X
(9) Brown, F. F.; Campbell, I. D.; Kuchel, P. W.; Rahenstein, D. L. FEBS. Lett.
____.__. __
1J77.R9 17-1fi (10) Hore, P. J. J. Magn. Reson. 1983,55,
283-300.
(11) Kalk, A,; Berendson, H.J.C. J. Reson. 1976,24,343-66.
Magn.
(12) Rahenstein, D. L.; Isab, A. A,; Brown,
D. W. J. Mngn. Reson. 1980,41,361-65. (13) Nicholson, J. K.; Buckingham, M. J.; Sadler, P. J. Biochem. J . 1983,211,60515. (14) Nicholson, J. K.; O'Flynn, M. P.; Sadler. P. J.; Macled, A. F.; Juul, S. M.; Sonksen, P. H. Biochem. J. 1984. 217,
Resonance Spectroscopy; Jackman. L. M ,Cotton. F.A.. Eds.; Academic Press New York, 1975: pp. 131-62 (21) Hahn. E. L. Phis. Re" 1950.80, 580Nudeor
Reson. 1979,36,281-86. (29) Nakashima. T. T.; Rabenstein, D. L. J . Magn. Reson. 1988,66,157-63. (30) Rabenstein, D. L.; Srivatsa, G. S.;Lee, R.W.K. J. Magn. Reson. 1987,71,175-79. (31) Bock, J. L. Clin. Chem. 1982,28,187371
(32i'F~sel,E,T.;Carr,J. M.; McDonagh,J. N.Engl. J. Med. 1986,315,1369-76. (33) Sim son, R.J.;Brindle, K. M.; Brown, F. F.; tampbell, I. D.; Foxall, D. L. Biochem. J . 1982,202,589-602. (34) Rabenstein, D. L.; Backs, S. J.; Isab, A. .. A. J . Am. Chem. Soe. 1981,103,283641.
(35) Rabenstein, D. L.; Isab. A. A,; Reid, R. S.Biochim. Biophys. Acta. 1982.696. 53-64. (36) Rabenstein, D. L.; Isab, A. A. Biochirn. Bio hys. Acta. 1982,722,374-84. (37) 8 hapman. B. E.; Beilharz, G. R.;York, M. J.; Kuchel, P. W. Bioehem. Biophys. Res. Commun. 1982.105.1280-87. (38) Kuchel, P. W..;Chapman, B. E. Biomed. Biochim. Actn. 1983, 42, 114349. (39) Rabenstein. D. L.; Isab, A. A,; Kadima, W.; Mohanakrishnan, P. Biochim. Biophys. Acta. 1983,762,531-41. (40) Beilharz, G. R.; Middlehurst, C. R.; Kuchel, P. W.; Hunt, G. E.; Johnson, G.F.S. AnaLBiochem. 1984,137,324-29. (41) Kuchel, P.W.; Hunt, G. E.; Johnson, G.F.S.; Beilbarz, G. R.; Chapman, 8 . E.; Jones, A. J.; Singh, B. S.J. AffectiveDisord. 1984,6,83-94. (42) Thorburn, D.R.;Kuchel, P. W. Eur. J. Biochem. 1985,150,371-86. (43) Guy, R. D.; Razi, M. T.; Rabenstein, D. L. J . Magn. Reson. 1986,66,434-44. (44) Rabenstein, D.L.; Arnold, A. P.; Guy, R.D. J . Bioinorg. Chem. l986,28,279-87. (45) Beilharz, G. R.;Middlehurst, C. R.; Kuchel, P. W.; Hunt, G. E.; Johnson, G.F.S. Aust. J.Exp. Biol. Med. Sci. 1986, 64,271-89. (46) Rabenstein! D. 4.; Mlllis, K. K., University of California, Riverside, unpublished results.
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e
Dallas L.Rabenstein (left)receiued a B.S. degree from t h e Uniuersity of Washington in 1964 and a Ph.D. from t h e University o f Wisconsin, Madison, in 1968. After a year as a lecturer at t h e Uniuersity of Wisconsin, Madison, and a year at Cheuron Research Co., he joined t h e faculty o f t h e Uniuersity of Alberta, Edmonton, Canada. I n I985 he accepted his present position as professor of chemistry at the Uniuersity o f California, Riverside. His research interests are in bioanalytical N M R spectroscopy, including t h e development o f new pulse Fourier transform methods and their application to t h e characterization o f peptides, proteins, biological fluids, and intact red blood cells and mast cells. Other research interests include t h e biological chemistry of sulfur and selenium and t h e chemistry of heavy metal toxicology. Kevin K. Millis (center) is a research assistant andgraduate student in analytical chemistry at the Uniuersity of California, Riverside, where he receiued a n M.S. degree. He receiued a B.S. degree in chemistry with a biochemistry emphasis from California State University a t Fullerton in 1986. His research interests are in analytical applications of N M R spectroscopy t o biochemical and medical problems. Erin J. S t r a w s (right) is a graduate student in analytical chemistry at t h e University of California, Riuerside. S h e receiued a B.A. degree in chemistry from Whittier College in 1986. Her research interests include bioanalytical NMR spectroscopy and the chemistry of sulfur in biological molecules.
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